Coupling mechanisms to activate store-operated and TRP channels

Abstract

Despite the precision with which spatial and temporal details of Ca 2+ signals have been resolved, a fundamental aspect of the generation of Ca2+ signals, the activation of "store-operated channels" (SOCs), remains a molecular and mechanistic mystery. The TRP family of receptor-operated channels share several operational parameters with SOCs and the question of whether activation of SOCs and TRP channels is mediated by the inositol-1,4,5-trisphosphate (InsP3) receptor (InsP3R) is examined. The permeant InsP3R-antagonist, 2-aminoethoxydiphenyl borate (2-APB) was previously reported to block SOCs and TRPs in an InsP3R-dependent fashion. Electroretinogram recordings of the light-induced current in Drosophila revealed that the TRP channel-mediated light response is inhibited by 2-APB. This action of 2-APB is likely to be InsP3R-independent since InsP3Rs are dispensable for the light response. We used a triple InsP3R knockout variant line of DT40 chicken B cells to further assess the role of InsP3Rs in SOC and TRP activation. 45Ca2+ flux analysis revealed that only the wild-type DT40 cells retain normal InsP3R-mediated, 2-APB-sensitive, Ca 2+ release. In intact cells, all parameters of Ca2+ store-function and coupling to activate SOCs were identical in DT40 wt and DT40InsP3R-k/o cells. Moreover, in both cell lines SOC-activation is completely blocked by 2-APB with identical kinetics of inhibition. We transiently transfected TRPC3 channels into the DT40 cells, and found that (a) endogenous B-cell receptors (BCR) coupled to phospholipase C-gamma (PLC-gamma); (b) exogenously expressed muscarinic receptors coupled to phospholipase c-gamma (PLC-gamma), and (c) the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG) activate the expressed TRPC3 channels in both DT40w/t and DT40InsP3R-k/o cells. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor U73122, and by wortmannin-induced PLC substrate depletion. However OAG-induced TRPC3 activation and store-operated channel activation remained unaffected under these conditions. We found that in the DT40 cells, 2-APB was a powerful InsP3R independent activator of store-operated Ca2+ entry between 1--10 muM. 2-APB activated authentic SOCs since the entry was selective for Ca2+ and highly sensitive to inhibition by La3+. With both w/t and InsP3R-k/o DT40 cells, the actions of 2-APB were restricted to SOCs in a store-coupled state. The results indicate that SOC and TRPC3 activation can occur independent of functional InsP 3Rs, and that in the DT40 cells TRPC3 channels are activated almost entirely by DAG following the stimulation of PLC-beta or -gamma. Furthermore, the biphasic effects of 2-APB reflect activation of authentic SOCs and 2-APB appears to modify SOCs by targeting the elusive coupling mechanism.